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Ke-Ke Zhang, Xiang-Jia Zhu, Yi Lu; Influence of α-crystallin Racemization on Lens Membrane Fluidity. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5583.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the influence of α-crystallin racemization on lens membrane fluidity using the mild heating model based on lens epithelial cells (LECs).
LECs (SRA01/04) heated at 500C were used as the mild heating model to simulate the ageing process in vitro. After incubation under 500C for 0min, 15min, 30min, 45min and 60min, α-crystallins in LECs were isolated using gel filtration chromatography and processed with acidolysis. The purified samples were treated with o-phtalaldehyde and N-tertbutyloxycarbonyl-L-cysteine for pre-column derivatization before racemization analysis using reversed-phase high performance liquid chromatography (RP-HPLC). To detect the α-crystallin racemization, we calculated the D/L (D/L amino-acid residual) ratio according to the peak area in each chromatogram. Laurdan staining and two-photon confocal microscopy were applied to analyze the lens membrane fluidity of each group with different heating time. Immunofluorescence staining were used to detect the expression and location of αA-crystallin (CRYAA) in LECs. The chaperone potential of a-crystallin under conditions of mild heating was also detected.
As to the mild heating model, racemization analysis on α-crystallin samples of LECs revealed an increased D/L ratio with the elongation of heating. The membrane fluidity of LECs also increased simultaneously along with heating, accompanied by racemized CRYAA gradually accumulated around the nucleus. Moreover, α-crystallin isolated from mild-heated hLECs showed a relatively lower protective effect according to the molecular chaperone function assessment.
Mild heating model on LECs was a potential method of simulating the ageing process and in vitro observation. Our study showed an increasing tendency of α-crystallin racemization with the passage of heating time. The increase of α-crystallin racemization may induce the increase of lens membrane fluidity and reduce its normal function as the molecular chaperone.
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